Over the past few decades there has been considerable development of highly sensitive and specific detection devices, particularly in the fields of biochemistry and molecular biology. Development has revolved around the use of time-honoured biomolecules such as enzymes, antibodies, oligonucleotides and other protein or DNA/RNA based compounds, which inherently possess remarkable recognition characteristics in biological systems. Recently, attention has turned to in situ detection technology, such as enzyme linked immunosorbent assays (ELISA) and microarrays. Progress has been hampered, however, because of the relative instability of these biomolecules in conditions outside of their native milieu, thus resulting in situations where reliability is compromised. In abnormal temperature and pH environments, proteins often degrade, losing their tertiary/quaternary structures and their inherent functionality. Fragmentation of nucleic acid probes and targets is typical, leading to false positive results. While “DNA chips” and other comparable microarray devices have only recently begun to achieve commercial success, they have been plagued by a lack of coherent and flexible detection criteria. Protein-based detection technology is still in a troubled infancy, hampered by logistical problems with the stabilization of the protein target to the array surface. Methods for the detection of biochemicals often lack sufficient sensitivity and specificity to discriminate between similarly structured entities. Furthermore, the methods used tend to be bulky, expensive, laborious and often involve dangerous chemicals for analysis.
Thus, there was a need to develop artificial recognition elements which could simulate the sensitive and specific detection capabilities of biomolecules. This need led to the discovery of molecular imprinting. Standard molecular imprinting is a process by which guest molecules (functional monomers or polymers) are allowed to self-assemble around a molecular target, conforming its shape into a recognition element, in relation to stearic interactions at binding sites corresponding to functional groups in the target molecule. The recognition elements form a binding cavity which is cross-linked into a matrix. The target molecule is removed, leaving behind a molecularly-imprinted polymer (MIP) complementary in shape and functionality to the target molecule, which will rebind chemical targets identical to the original molecular target. The MIP functions like a lock that is only compatible with the correct key. Molecular imprinting has been used in column chromatography and silica gel applications (Sulitzky et al., Macromolecules 2001) and for the synthesis of polymeric materials that mimic biological receptors.
Molecular recognition between a chemical target and its corresponding MIP requires high order discrimination at the binding sites; this can happen only if the binding sites of the MIP and chemical target complement each other in size, shape, orientation, and chemical functionality. MIPs possess high affinity and selectivity (similar to natural receptors and antibodies), are uniquely stable as compared to natural biomolecules, are relatively simple to prepare, and easily adaptable to different applications (Piletsky et al., 2001).
Early work in molecular imprinting, particularly by Wolff et al., provided a guest molecule-molecular target interaction whereby aggregates in solution are maintained by reversible covalent bonds. Recent development by Mosbach and others has focussed on the “supra-molecular assembly” approach, whereby the pre-arrangement between the molecular target and the guest molecule is formed by non-covalent or metal co-ordination interactions.
The guest molecule-molecular target complex is then copolymerized with a cross-linker, leading to a highly cross-linked macroporous polymer with the imprint molecules in a sterically-fixed arrangement. This forms a highly-specific recognition element capable of recognizing a molecular target. The cross-linker forms an insoluble polymer with substantial rigidity. Since a very high degree of cross-linking (70-90%) is necessary for achieving specificity, only a limited number of cross-linkers have been utilized. Further, introducing external cross-linkers to the imprinting process can potentially hinder MIP formation if cross-linking at undesired locations within the MIP occurs. Several different cross-linkers have been tried with different degrees of success. Originally, isomers of divinylbenzene were used for cross-linking of styrene and other functional monomers into polystyrenes. Later, it was found that acrylic or methacrylic based systems could be prepared with much higher specificity. In addition to cross-linkers which are non-polar or weakly polar in nature, cross-linkers containing functional groups have been studied (Ramström, 1996).
Typically, binding cavities comprising the polymerized guest molecule have been cross-linked into three-dimensional “blocks” which recognize targets which become embedded within their structure. The block of polymer is then crushed, ground and filtered to produce appropriate particle sizes. The particles generated are irregular and some binding sites may be destroyed by the grinding process. The three-dimensional nature of these materials has an effect on the porosity of the matrix, meaning that adsorption and desorption kinetics play a major role in their effectiveness. Moreover, and because no direct analytical technique (such as surface plasmon resonance (SPR), cantilever or evanescent-based techniques) can probe the adsorption of the target molecules, multiple chemical steps, such as extraction, concentration, labelling etc., are required. This series of steps has hindered the use of MIPs outside of the lab setting. Such three-dimensional polymers have been described by Mosbach and in U.S. Pat. No. 6,310,110, issued to Markowitz et al.
Molecular imprinting had been typically carried with the molecular target as a free, unbound moiety in solution target free in solution. Because the molecular targets are not fixed to a solid surface, the quality of the MIPs can be compromised, resulting in binding sites which are heterogeneous in their orientation, shape and affinity for/accessibility to the target molecule. U.S. Pat. No. 5,630,978, issued to Domb, discloses an in vitro method for the preparation of mimics of drugs and other biologically active molecules.
Recent progress has been made in surface immobilization of the molecular target on a solid support. Friggeri et al. (Angew. Chem. Int. Ed. 2001; 40(24):4729-4731) describe a novel, solution-to-surface imprinting method based on the different higher-order conformations adopted by boronic acid appended poly-L-lysine. A molecular imprinted surface using boronic acid for binding sugars has been described by Miyahara and Kurihara (Chemistry Letters 2000; 1356), and using theophylline on aminopropyl-derivatized silica gel has been described by Yilmaz et al. (Angew. Chem. Int. Ed. 2000; 39(12):2115-2118).
PCT application WO 01/90228 (Mosbach et al.) discloses a molecular imprinting technique using immobilized templates for imprinting. This process ensures that the imprinted polymers produce uniform and structurally well-defined binding sites. The binding sites are located at or close to the surface of the polymer. This method uses a less reliable three-dimensional MIP which must be degraded with harsh chemicals (such as hydrofluoric acid) and loaded into a chromatography column for detection of bound targets. Similar imprinting methods are described in U.S. Pat. No. 6,489,418 and U.S. Pat. No. 6,127,154, both also issued to Mosbach.
Shi et al. (Nature 1999; 398:593-7) describe a method for synthesizing molecular target-imprinted surfaces for protein recognition. The target protein used is adsorbed to a mica thin layer. The “cavity” is formed from a saccharide which coats the protein. A plasma film overlays the cavity and is attached to a solid support. Although the system shows differential recognition of similar elements through strict conformational structures, its use appears to be limited to biological-based target proteins (such as antibody IgG and albumin). A method for making imprinted protein recognition sites for use in synthetic enzyme and antibody applications is disclosed in U.S. Pat. No. 5,110,833, issued to Mosbach. PCT application WO 01/88960 (Polyani et al.) describes a process for forming an imprinted pattern of adsorbed molecules on a solid surface.
Soft lithography represents a new alternative for fabricating MIP microstructures on two-dimensional substrates, rather than in the traditional three-dimensional matrix. The original version of this technology employed an elastomeric stamp (or mold), typically made of poly(dimethylsiloxane) (PDMS), to pattern a wide variety of materials such as self-assembled monolayers (SAMs), organic polymers, colloids, inorganic solids, proteins, and cells. The stamp is then placed in contact with a solid substrate, such as a gold coated glass slide. Although standard soft lithography is a relatively simple technique which requires no special equipment and can routinely be performed in the typical laboratory, deformations and distortions can be readily introduced due to the flexibility of the elastomeric stamp leading to errors in the replicated pattern.
A technique for fabricating MIP microstructures on silicon wafers using soft lithography has been described by Yan and Kapua (Anal. Chim. Acta 2001; 435:163-67). A PDMS stamp is contacted with a wafer creating microchannel structures on its surface. Formation of the MIP is achieved by fluidic means whereby the functional monomers are injected into the microcapillaries. Once the stamp is removed, the micro-channels are imprinted with MIPs. This method relies on capillarity to fill the channels with the polymer and bind target molecules to the MIPs. The method is somewhat impractical, however, as the detection of bound targets requires the use of lab-based technology such as column chromatography, HPLC, or GC/MS.
Methods for synthesizing MIPs have usually been limited to one molecular target of interest. Because of this, the use of molecular imprinting technology in a wide assortment of applications has been restricted. This is particularly crucial for the detection of biochemical families which as an ensemble may be responsible for the accurate diagnosis of certain diseases, such as, for example, the levels of dopamine in Parkinson's Disease or in the monitoring of different troponin isoforms in myocardial infarction.
The detection of hazardous and potentially lethal chemicals has become increasingly essential with continued threats of bioterrorism and the presence of harmful pollutants in the atmosphere. Detection and quantification of these toxic compounds at very low levels are critical, and must be done quickly so that appropriate precautions can be taken and/or emergency treatment can be provided. Although a variety of physical, chemical and biological techniques have been investigated, few detectors are small and inexpensive enough to be used in situ, such as in military or environmental applications. In addition, methods for the unambiguous detection and quantification of specific gasses usually involves separate sampling and analysis steps using complex and expensive equipment such as gas chromatography/mass spectrometry and HPLC. Devices based on this technology are not portable and are expensive and/or require extensive analysis procedures making them undesirable for real-time field analysis. Testing can take up to 24 hours to perform and typically lack specificity and simplicity. Existing detection devices can yield high false positive rates, particularly in attempts to distinguish nerve gasses (such as sarin) from other organophosphorous compounds which can be found in pesticides and insecticides.
With the recent surge in interest and necessity for the detection of nerve gasses and other potentially lethal chemicals, attempts have been made to manufacture sensitive chemical detection devices. However, very few use direct quantification of toxin of interest. A silicon chip sensor, designed by Trogler and colleagues at the University of San Diego for the U.S. Army (220th meeting of the American Chemical Society, 2000), uses a molecular catalyst to break the phosphorous-fluorine bond in “G”-type nerve agents. The product of this reaction, hydrogen fluoride, is detected with a silicon interferometer. The device, therefore, uses an indirect method of nerve gas detection and quantification. Hazardous chemical sensors are described in US Application 2003/0027936 (Murray et al.) and by Jenkins et al. (Anal Chem. 1999; 71(2):373-8).
It is, therefore, desirable to provide a novel method of producing a molecularly-imprinted chemical detection device.